Several strains of yeast are known to excrete alpha, omega-dicarboxylic acids as a byproduct when cultured on alkanes or fatty acids as the carbon source. In particular, yeast belonging to the genus Candida, such as C. albicans, C. cloacae, C. guillermondii, C. interinedia, C. lipolytica, C. maltosa, C. parapsilosis and C. zeylenoides are known to produce such dicarboxylic acids (Isamu Shiio and RyousukeUchio, Agr. Biol. Chem. 1971, 35: 2033-2042). In C. tropicalis, the first step in the omega-oxidation pathway is catalyzed by a membrane-bound enzyme complex (omega-hydroxylase complex) comprised of a cytochrome P450 monooxygenase and an NADPH-cytochrome reductase. This hydroxylase complex is responsible for the primary oxidation of the terminal methyl group in alkanes and fatty acids (Michele Gilewicz, Marcelle Zacek, Jean-Claude Bertrand, and Edgard Azoulay, Can. J Microbiol., 1979, 25, 201).
It has been established that hydrocarbon substrates are enzymatically oxidized in the yeast microsomes. Following transport into the cell, n-alkane substrates for example, are hydroxylated to fatty alcohols by a specific cytochrome P450 system. Two further oxidation steps, catalyzed by alcohol oxidase and aldehyde dehydrogenase, lead to the corresponding fatty acid. The fatty acids can be further oxidized through the same pathway to the corresponding dicarboxylic acid (Colin Ratledge, J. Am. Oil Chem. Soc., 1984, 61(2), 447-453). The di-terminal oxidation, leads to dicarboxylic acid production, from aliphatic hydrocarbons by yeasts (Shigeo Ogino, Keiji Yano, Gakuzo Tamura and Kei Arima, Agri. Biol. Chem., 1965, 29(11), 1009-1015).
The omega-oxidation of fatty acids proceeds via the omega-hydroxy-fatty acid and its aldehyde derivative, to the corresponding dicarboxylic acid without the requirement for CoA activation. However, both fatty acids and dicarboxylic acids can be degraded, after activation to the corresponding acyl-CoA ester, through the β-oxidation pathway in the peroxisomes (Atsuo Tanaka and saburo Fukui, In: The Yeasts, Vol. 3, Metabolism and physiology of Yeasts, Edited by A. H. Rose and J. S. Harrison, 2nd Edition, Academic Press, Harcourt Brace Jovanovich, Publishers), leading to chain shortening. In yeast, beta-oxidation takes place solely in the peroxisomes (Mitsuyoshi Ueda, Kazunori Yamanoi, Tadashi Morikawa, Hirofumi Okada and Atsuo Tanaka, Agr. Biol. Chem., 1985, 49, 1821-1828).
Dicarboxylic acids produced through fermentation by most yeasts, including C. tropicalis, are often shorter than the original substrate by one or more pairs of carbon atoms and mixtures are common (Shigeo Ogino, Keiji Yano, Gakuzo Tamura and Kei Arima, Agr. Biol. Chem., 1965, 29(11), 1009-1015., Shio and Uchio, Agr. Biol. Chem., 1971, 35(13), 2033-2042). These undesirable by-products are often associated with biological production of dicarboxylic acids.
It is known that the formation of dioic acids can be substantially increased by the use of suitable mutants (RyousukeUchio and Isamu Shiio, Agri. Biol. Chem., 1972, 36(3), 426-433). The wild-type yeasts produce little if any dicarboxylic acid. Often, mutants partially defective in their ability to grow on alkane, fatty acid or dicarboxylic acid substrates demonstrate enhanced dicarboxylic acid yields. However, these mutants have not been characterized beyond their reduced ability to utilize these compounds as a carbon source for growth. In all likelihood, their ability to produce dicarboxylic acids is enhanced by a partial blockage of the beta-oxidation pathway. Furthermore, compounds known to inhibit beta-oxidation (i.e. acrylate) also result in increased dicarboxylic acid yields.
In addition, the use of such a mutant should prevent the undesirable chain modifications associated with passage through beta-oxidation, such as unsaturation, hydroxylation, or chain shortening.
Many organisms carry out the transformations, including Cryptococcus neoformans and Pseudomonas aeruginosa, Corynebacterium sp., and at least two strains of Candida, that is C. cloacae and C. tropicalis. (Kenneth D. Green, Michael K. Turner, Johm M. Woodley, Enzyme and Microbial Technology, 2000, 27, 205-211).
The work with the bacterial cells has used wild-type organisms, in which solvents, detergents, and immobilization may all give improved conversions (E. C. Chan and J. Kuo, Biotransformation of dicarboxylic acid by immobilized Cryptococcus cells. Enzyme Microb Technol, 1997, 20, 585-589). In contrast, the work with the yeast strains has usually used mutants of Candida tropicalis in which the β-oxidation of fatty acids is impaired. Engineered strains of C. tropicalis that lack several key enzymes of β-oxidation are particularly effective catalysts for these oxidations. This directs the metabolic flux toward ω-oxidation, and the n-alkanes are more efficiently converted to the corresponding dioic acids.